Abstract As multidrug-resistant (MDR) bacteria are increasingly difficult to treat and are associated with greater morbidity and mortality, we are facing a global health crisis. Countermeasures that do not select for drug resistance are urgently needed. The overall goal of this research program is to develop novel immune-based preventive strategies against major MDR pathogens in developing and developed countries. In this project we aim to develop novel probiotic yeast-based immune-interventions against Clostridium difficile and Campylobacter jejuni, the major causes of bacterial diarrheal and intestinal inflammatory diseases worldwide. We have developed a novel platform technology against these enteric pathogens by engineering a probiotic yeast, Saccharomyces boulardii, to secrete multi-specific, single-domain antibodies directly targeting bacterial virulence factors at the intestinal site of infection. Through our previously funded Centers of Excellence for Translational Research (CETR) project, we have applied this technology to target C. difficile, and have already generated a prototype yeast strain that constitutively secretes a tetra-specific, single-domain-antibody (VHH) fusion which neutralizes the two major C. difficile enterotoxins TcdA and TcdB. A recently completed proof-of- principle study has shown that oral administration protected mice from both primary and recurrent C. difficile infection. Building upon these highly encouraging results, we propose to: 1) generate lead S. boulardii strains secreting multi-specific antibodies against bacterial colonization factors of C. difficile and C. jejuni; 2) preclinically evaluate the efficacy of the lead strains on reducing bacterial colonization and preventing disease in animal models; and 3) develop a formulation for drying and encapsulating these lead yeast strains that is compatible for future use in humans. We envision that our future final products for human use will be enteric coated capsules containing dried yeast strains, similar to those probiotic S. boulardii products sold over-the- counter. With the completion of proposed translational activities, we expect to generate a final improved product of engineered yeast strains to prevent C. difficile disease and a prototype product to prevent C. jejuni disease. Successful preclinical validation of the lead candidates will also allow further development and large- scale efforts for eventual commercialization of these products against C. difficile and C. jejuni diseases, for which we currently have no effective preventative measures.